CN102989000A - Process for preparing purified drug conjugates - Google Patents

Process for preparing purified drug conjugates Download PDF

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Publication number
CN102989000A
CN102989000A CN2012103082004A CN201210308200A CN102989000A CN 102989000 A CN102989000 A CN 102989000A CN 2012103082004 A CN2012103082004 A CN 2012103082004A CN 201210308200 A CN201210308200 A CN 201210308200A CN 102989000 A CN102989000 A CN 102989000A
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antibody
connector
mixture
cytotoxic agent
purification
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CN102989000B (en
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戴勇
王勇
晋圣瑾
D·H·梅舒拉姆
G·W·阿姆弗莱特
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Immunogen Inc
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Immunogen Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6867—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from a cell of a blood cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6807—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense
    • A61K47/6809—Antibiotics, e.g. antitumor antibiotics anthracyclins, adriamycin, doxorubicin or daunomycin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2839—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2884—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD44
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification

Abstract

The invention provides a process for preparing a cell-binding agent chemically coupled to a drug. The process comprises covalently attaching a linker to a cell-binding agent, a purification step, conjugating a drug to the cell-binding agent and a subsequent purification step.

Description

The method for preparing maytansinoid antibody conjugates
The application is that application number is 200680034242.6, the applying date is on August 14th, 2006, denomination of invention is divided an application for the patent application of " method for preparing maytansinoid antibody conjugates ".
Technical field
The present invention relates to prepare the method for suitable high-purity and the conjugate of stability, wherein said conjugate comprises the Cell binding agent with the pharmaceutical chemistry coupling.
Background technology
Along with targeting more effectively and kill the drug development of cancerous cell, treatment of cancer makes marked progress.For this purpose, cell surface receptor and antigen that research worker utilizes cancer cell selectivity to express are developed the medicine based on antibody, and described antibody is combined with tumour-specific or tumor related antigen.In this, with the cytotoxicity molecule for example antibacterial be connected with phytotoxin radionuclide be connected chemotherapeutic agent and monoclonal antibody chemistry and be connected, described monoclonal antibody be combined with tumour-specific or tumor related antigen (referring to, for example, International Patent Application WO 00/02587, WO 02/060955 and WO 02/092127, United States Patent (USP) 5,475,092,6,340,701 and 6,171,586, U.S. Patent Application Publication No. 2003/0004210A1, with Ghetie etc., J.Immunol.Methods, 112:267-277 (1988)).Usually this compounds is called toxin, radionuclide and medicine " conjugate ".Usually also they are called immunoconjugates, radioimmunity conjugate and immunotoxin.Be combined with tumor cell and discharge medicine or/and when activating the cytotoxic activity of medicine, the Cancer cell is killed at drug conjugate.Therefore the selectivity that drug conjugate provides makes and Normocellular toxicity is dropped to minimum, has improved the toleration of medicine in the patient.
Previous described with antibody and the cytotoxic agent that contains sulfydryl for example the maytansinoid method of puting together (referring to, for example, United States Patent (USP) 5,208,020,5,416,064 and 6,441,163).For example, United States Patent (USP) 5,208,020 and 5,416,064 discloses the method for Dispersal risk-maytansinoid conjugate, wherein at first use heterobifunctional agent (for example United States Patent (USP) 4,149,003,4,563,304 and U.S. Patent Application Publication No. 2004/0241174A1 described in) modified antibodies.United States Patent (USP) 5,208,020 and 5,416,064 has further described modified antibody and the excessive cytotoxic agent that contains sulfydryl in the puting together of pH 7, with by Sephadex TMG25 chromatographic column purification.Also described with size exclusion chromatography (SEC) (SEC) antibody purification-drug conjugate (referring to, for example, Liu etc., Proc.Natl.Acad.Sci (USA), 93:8618-8623 (1996) and Chari etc., Cancer Research, 52:127-131 (1992)).
The previous method of describing for the preparation of antibody-drug conjugates is complicated, because the burdensome step that they are used to carry out or produce immunoconjugates hinders, with comparing that the best is expected, described immunoconjugates purity is lower or more unstable.For example, pH be between 6.0～6.5 to put together for producing pure and stable conjugate be not best.In addition, conjugation reaction under these conditions causes needs to use excessive time and materials normally slowly with inefficient.
Changing or eliminate one or more preparation processes, and do not damage product quality for example purity and/or stability, will be desirable.Further it is desirable to, have except described other purification those up to now and select, will more effective because some use the selection of some combination of Cell binding agent, connector and medicine than what use other.
Described in view of preamble, there is in the art following demand: develop the method that improved preparation has suitable high-purity and has simultaneously the Cell binding agent-drug conjugate composition of larger stability.The invention provides such method.According to explanation of the present invention provided herein, these and other advantage of the present invention and other inventive features will be obvious.
Summary of the invention
The invention brief overview
The invention provides a kind of method for preparing the conjugate of suitable high-purity and stability, this conjugate comprises the Cell binding agent with the pharmaceutical chemistry coupling.The method comprises that (a) contacts with the Cell binding agent with difunctional cross-linking reagent, so that connector and Cell binding agent are covalently bound, thereby prepare the first mixture, this mixture comprises the Cell binding agent that combines connector on it, (b) make the first mixture stand tangential flow filtration, adsorption charomatography, adsorption filtration, selective precipitation or their combination, thereby combine the first mixture of the Cell binding agent of connector on its of preparation purification, (c) by being about 4 to about 9 solution, its Cell binding agent and medicine that combines connector to be reacted at pH, to prepare the second mixture, this mixture comprises (i) by the Cell binding agent of connector and pharmaceutical chemistry coupling, (ii) free drug and (iii) byproduct of reaction, the Cell binding agent that combines connector on it is puted together, and (d) make the second mixture stand tangential flow filtration, adsorption charomatography, adsorption filtration, selective precipitation or their combination, with purification from other composition of the second mixture by the Cell binding agent of connector and pharmaceutical chemistry coupling, thereby the preparation purification the second mixture.
Detailed Description Of The Invention
The invention provides a kind of method for preparing the Cell binding agent-drug conjugate of suitable high-purity and stability.Such compositions is because high-purity and the stability of this conjugate can be used for treating disease.Comprising the Cell binding agent for example for example is described among the U.S. Patent Application Publication No. 2004/0241174A1 with the compositions of the antibody of medicine such as maytansinoid chemical coupling.In the present context, quite high-purity is considered to: (a) greater than 90%, being preferably greater than 95% conjugate kind is monomer, and/or (b) in the conjugate prepared product free levels of drugs less than 2% (with respect to total medicine).
In this respect, method of the present invention comprises that (a) is with difunctional cross-linking reagent modified cells bonding agent, so that connector and Cell binding agent are covalently bound, thereby prepare the first mixture, this mixture comprises the Cell binding agent that combines connector on it, (b) make the first mixture stand tangential flow filtration, adsorption charomatography, adsorption filtration, selective precipitation or their combination, it combines the Cell binding agent of connector with purification from other composition of the first mixture, thereby combine the first mixture of the Cell binding agent of connector on its of preparation purification, (c) by being about 4 to about 9 solution, its Cell binding agent and medicine of combining connector to be reacted at pH, to prepare the second mixture, this mixture comprises (i) by the Cell binding agent of connector and pharmaceutical chemistry coupling, (ii) free drug and (iii) byproduct of reaction, the Cell binding agent that combines connector on it is puted together, (d) make the second mixture stand tangential flow filtration, adsorption charomatography, adsorption filtration, selective precipitation or their combination are to remove the non-medicine of puting together, reactant and by-product and the Cell binding agent-drug conjugate that obtains suitable purification.
Preferably, in purification step, utilize tangential flow filtration (TFF is also referred to as cross-flow filtration, ultrafiltration and diafiltration) and/or adsorption charomatography resin.Yet, when TFF is used for first purification step (step b), the pH that puts together application in (step c) is 6.0-6.5, and when using the adsorption charomatography resin in second purification step (steps d), preferred adsorption charomatography resin is nonionic exchange resin.In another preferred embodiment, TFF is used for two purification steps, or the adsorption charomatography resin is used for two purification steps.Alternative ground, the adsorption charomatography resin is used for first purification step, and TFF is used for second purification step.The combination of TFF and adsorption charomatography resin also can be used in first and/or second purification step.
Any suitable TFF system be can utilize, Pellicon type system (Millipore, Billerica comprised, MA), Sartocon Cassette system (Sartorius AG, Edgewood, NY) and (the Pall Corp. of Centrasette type system, East Hills, NY).
Can utilize any suitable adsorption charomatography resin.Preferred adsorption charomatography resin comprises the resin for hydroxyapatite chromatography method, dewatering electric charge inducing color chromatogram method (HCIC), hydrophobic interaction chromatography (HIC), ion exchange chromatography, hybrid ion exchange chromatography, immobilized metal affinity chromatography (IMAC), dye ligand chromatography, affinity chromatography, reverse-phase chromatography and combination thereof.The example of applicable hydroxyapatite resin comprises ceramic hydroxyapatite (I type and II type CHT, Bio-Rad Laboratories, Hercules, CA), HA Ultrogel hydroxyapatite (Pall Corp., East Hills, NY) and ceramic fluor-apatite (I type and II type CFT, Bio-RadLaboratories, Hercules, CA).The example of applicable HCIC resin is MEPHypercel resin (Pall Corp., East Hills, NY).The example of applicable HIC resin comprises that Butyl-Sepharose, Hexyl-Sepaharose, Phenyl-Sepharose and Octyl Sepharose resin are (all from GE Healthcare, Piscataway, NJ) and Macro-prep Methyl and Macro-Prep t-Butyl resin (Biorad Laboratories, Hercules, CA).The example of applicable ion exchange resin comprises that SP-Sepharose, CM-Sepharose and Q-Sepharose resin are (all from GE Healthcare, Piscataway, NJ) and Unosphere S resin (Bio-Rad Laboratories, Hercules, CA).The example of the hybrid ion exchange resin that is fit to comprises Bakerbond ABx resin (JTBaker, Phillipsburg NJ).The example of applicable IMAC resin comprises Chelating Sepharose resin (GE Healthcare, Piscataway, NJ) and Profinity IMAC resin (Bio-Rad Laboratories, Hercules, CA).The example of suitable dyes part resin comprises Blue Sepharose resin (GE Healthcare, Piscataway, NJ) and Affi-gelBlue resin (Bio-Rad Laboratories, Hercules, CA).The example of the affine resin that is fit to (for example comprises Protein A Sepharose resin, MabSelect, GE Healthcare, Piscataway, NJ), wherein the Cell binding agent is antibody, with the affine resin of agglutinin Lentil Lectin Sepharose resin (GE Healthcare for example, Piscataway, NJ), wherein suitable Lectin Binding Sites is carried in the Cell binding agent.The antibody special to the Cell binding agent can be used in alternative ground.Such antibody can be fixed on, for example, and on the Sepharose 4Fast Flow resin (GE Healthcare, Piscataway, NJ).The example of the reversed-phase resin that is fit to comprises C4, C8 and C18 resin (Grace Vydac, Hesperia, CA).
The method according to this invention prepares the first mixture, and it comprises the Cell binding agent that combines connector on it, and reactant and other by-product.By making the first mixture stand purification process, the Cell binding agent that purification is modified from reactant and by-product.In this, can use tangential flow filtration (TFF) and for example come purification the first mixture based on methods of tangential flow filtration, adsorption charomatography, adsorption filtration or selective precipitation or any other applicable purification process and their combination of film.This first purification step provides the first mixture of purification, in other words, compares with the first mixture before the purification according to the present invention, has increased the concentration and the amount that has reduced unconjugated difunctional cross-linking reagent of the Cell binding agent that combines connector on it.
Combine first mixture of Cell binding agent of connector with its that obtains purification at purification the first mixture after, by being about 4 to about 9 solution, its Cell binding agent and medicine of combining connector to be reacted at pH, therefore prepare the second mixture, it comprises (i) Cell binding agent by connector and pharmaceutical chemistry coupling, (ii) free drug and (iii) byproduct of reaction, and the Cell binding agent that combines connector on it is puted together.Although conjugation reaction is carried out to about pH 9 at about pH 4, but reaction is about 6 or lower or be about 6.5 or larger carrying out at pH at pH preferably, most preferably be about 4 to about 6 or be about 6.5 to about 9 at pH at pH, especially pH be 4 to less than 6 or at pH greater than 6.5 to 9.Be about 6.5 or during larger carrying out when puting together step at pH, some medicines that contain sulfydryl form by disulfide bond and can tend to dimerization.In this case, consider high efficiency reaction, may need from reactant mixture, to remove trace metal and/or oxygen, and optional add antioxidant or use has the connector of more reactive leaving group, or add the medicine that surpasses an aliquot.
Randomly, can omit the purification of the Cell binding agent of modification.Under situation like this, medicine can with cross-linking reagent simultaneously or after time point is for example adding cross-linking reagent after a while 1,2,3 or joined in more hours in the Cell binding agent.
The inventive method can be chosen wantonly and comprise that sucrose is added in used puting together in the step in the inventive method, to increase dissolubility and the response rate of Cell binding agent-drug conjugate.Desirably, the concentration of sucrose adding is that about 0.1% (w/v) is to about 20% (w/v) (for example about 0.1% (w/v), 1% (w/v), 5% (w/v), 10% (w/v), 15% (w/v) or 20% (w/v)).Preferably, the concentration of sucrose adding is that about 1% (w/v) is to about 10% (w/v) (for example about 2% (w/v), about 4% (w/v), about 6% (w/v) or about 8% (w/v)).In addition, conjugation reaction also can comprise the interpolation buffer agent.Can use any applicable buffer agent known in the art.Applicable buffer agent comprises for example citrate buffer agent, acetate buffer, succinate buffer agent and phosphate buffer.
After puting together step, make the second mixture stand purification step.In this, the second mixture can be used tangential flow filtration (TFF) and for example carry out purification based on methods of tangential flow filtration, adsorption charomatography, adsorption filtration, selective precipitation or any other applicable purification process and their combination of film, and these are all mentioned in this article.This second purification step provides the second mixture of purification, in other words, compare with the second mixture before the purification according to the present invention, increased concentration and the amount that has reduced one or more other compositions in the second mixture by the Cell binding agent of connector and pharmaceutical chemistry coupling.
The Cell binding agent can be any suitable material with Cell binding, and described cell is general and be preferably zooblast (for example people's cell).The Cell binding agent is preferably peptide or polypeptide.Applicable Cell binding agent comprises any other material or the molecule of target molecule on for example antibody (for example, monoclonal antibody and its segment), lymphokine, hormone, somatomedin, nutrition transport molecules (for example transferrins) and the specific binding cell surface.
Term used herein " antibody " refers to any immunoglobulin, and any immunoglobulin fragments is Fab, F (ab') for example 2, dsFv, sFv, double antibody and three chain antibodies, or immunoglobulin chimeric body, they can be combined (for example, they contain complementary determining region (CDR)) by the antigen on cell surface.Any applicable antibody can be used as the Cell binding agent.The selection that it will be appreciated by the skilled addressee that suitable antibody will be depended on will be by the cell mass of targeting.In this, the type of selective expression's cell surface molecule (being antigen) and number will determine selection for the suitable antibody of the present composition in specific cells group (general and be preferably diseased cells group).For extensive multiple cell type (comprising tumor cell type), the cell surface expression characteristic is known, if perhaps do not know, can use conventional molecular biology and tissue chemical technology and measure.
Antibody can be polyclone or monoclonal antibody, but most preferably is monoclonal antibody." polyclone " used herein antibody refers to the antibody molecule of heterogeneous population, generally is included in the serum of immune animal." monoclonal " antibody refers to the antibody molecule of homogenizing colony, and it is specific to specific antigen.Monoclonal antibody generally produces by the single clone of bone-marrow-derived lymphocyte (" B cell ").Use various technology well known by persons skilled in the art and comprise the standard hybridoma technology, can obtain monoclonal antibody (referring to, for example, And Milstein, Eur.J.Immunol, 5:511-519 (1976), Harlow and Lane (editor), Antibodies:A Laboratory Manual, CSH Press (1988), and C.A.Janeway etc. (editor), Immunobiology, the 5th edition, Garland Publishing, New York, NY (2001)).Briefly, the hybridoma method of manufacture order clonal antibody generally comprises to any applicable animal (general and be preferably mice) injections of antigens (i.e. " immunogen ").Put to death subsequently animal, will merge from the isolated B cell of its spleen and human myeloma cell.Produce hybrid cell (i.e. " hybridoma "), it is in external ad infinitum hypertrophy and secrete continuously and have the specific high titre antibody of expectation.Any suitable method known in the art can be used for identifying producing to have the hybridoma of expecting specific antibody.Such method comprises for example enzyme-linked immunosorbent assay (ELISA), western blot analysis and radioimmunoassay.Screening hybridoma colony is to separate each clone, and each clone's secretion is for the monospecific antibody kind of antigen.Because the clone that each hybridoma is served as reasons and produced with single B cell fusion, all antibody molecules of its generation structurally are identical, comprise their antigen-binding site and isotype.Also can use other suitable technology manufacture order clonal antibody, comprise the EBV-hybridoma technology (referring to, for example, Haskard and Archer, J.Immunol. Methods, 74 (2): 361-67 (1984), and Roder etc., Methods Enzymol, 121:140-67 (1986)), the phage vector expression system (referring to, for example, Huse etc., Science, 246:1275-81 (1989)) or comprise antibody fragment such as Fab and scFv (strand variable region) phage display library (referring to, for example, United States Patent (USP) 5,885,793 and 5,969,108, and International Patent Application WO 92/01047 and WO 99/06587).
Monoclonal antibody can be separated from any suitable animal or be produced therein, but preferably in mammal, more preferably in mice or people, and most preferably produces in the people.The method that produces antibody in mice is well known to those skilled in the art, and is described in herein.As for people's antibody, it will be appreciated by the skilled addressee that polyclonal antibody can separate from the experimenter's that crosses with suitable antigen inoculation or immunity serum.Alternative ground, the known technology that for example produces people's antibody in the mice the non-human animal by adaptive change can produce people's antibody (referring to, for example, United States Patent (USP) 5,545,806,5,569,825 and 5,714,352, and U.S. Patent Application Publication No. 2002/0197266A1).
Although be the ideal chose that the human treatment uses, people's antibody, especially human monoclonal antibodies more is difficult to produce than mouse monoclonal antibody usually.Yet mouse monoclonal antibody causes quick host's antibody response when using to the people, and this can reduce treatment or the diagnostic potential of antibody drug conjugate.In order to prevent these complication, preferred monoclonal antibody is not thought " exotic " by human immune system.
For this purpose, phage display can be used for producing this antibody.In this, application standard molecular biology and recombinant DNA technology, can produce the antigen of encoding antibody in conjunction with the phage library in variable (V) territory (referring to, for example, Sambrook etc. (editor), Molecular Cloning, A Laboratory Manual, the third edition, Cold Spring Harbor Laboratory Press, New York (2001)).For the antigen of specific binding expectation, select coding to have the phage of the specific variable region of expectation, and rebuild the fully human antibodies that comprises selected variable domain.The nucleotide sequence of coding reshaped antibody is introduced the myeloma cell that suitable cell line is produced as being used for hybridoma so that the people's antibody with monoclonal anti bulk properties by this emiocytosis (referring to, for example, Janeway etc., above, Huse etc., above with United States Patent (USP) 6,265,150).Alternative ground, monoclonal antibody can be that genetically modified mice produces by and light chain immunoglobulin gene heavy to specific human.Such method is known in the art, and is described in for example United States Patent (USP) 5,545,806 and 5,569,825 and Janeway etc., above.
Most preferably, antibody is humanized antibody." humanization " used herein antibody is complementary determining region (CDR) (it forms the antigen coupling collar of the antibody) antibody of grafting on the framework of human antibody molecules of wherein mouse monoclonal antibody.Because the similarity of mice and people's antibody framework, the monoclonal antibody that this method recognized in the art produces is that antigen is identical with people's antibody, but can with the mouse monoclonal antibody of generation CDR sequence in conjunction with identical antigen.The method of producing humanized antibody is well known in the art, and be described in detail in such as Janeway etc., above, United States Patent (USP) 5,225,539,5,585,089 and 5,693,761, european patent number 0239400B1, and in the British Patent No. 2188638.Also can use United States Patent (USP) 5,639,641 and Pedersen etc., J.Mol Biol, the antibody surface reconstruction technology described in the 235:959-973 (1994) produces humanized antibody.Although antibody used in the conjugate of the present composition most preferably is Humanized monoclonal antibodies, aforesaid human monoclonal antibodies and mouse monoclonal antibody are also within the scope of the invention.
Has at least one antigen-binding site and therefore identification and in conjunction with the antibody fragment that is positioned at lip-deep at least one antigen of target cell or receptor, also within the scope of the invention.In this regard, the proteolytic cleavage of complete antibody molecule can produce the Multiple Antibodies segment, and described antibody fragment keeps the ability of identification and conjugated antigen.For example produce three segments with papain limited digestion antibody molecule is general, wherein two are identical and are called the Fab segment, because their keep the antigen-binding activity of maternal antibody molecule.With two kinds of antibody fragments of the normal generation of pepsin cracking antibody molecule, wherein therefore a kind of two antigen brachium conjunctivums that kept this antibody molecule are called F (ab') 2Segment.With dithiothreitol, DTT or mercaptoethylmaine reduction F (ab') 2Segment produces the segment that is called the Fab' segment.Use conventional recombinant DNA technology, can produce strand variable region segment (sFv) antibody fragment, it is comprised of the Fab segment of truncate, described Fab segment comprise the heavy chain of antibody that is connected with the V territory of light chain of antibody through synthetic peptide variable (V) territory (referring to, for example, Janeway etc., above).Similarly, by recombinant DNA technology, can prepare the stable variable region segment (dsFv) of disulphide (referring to, for example, Reiter etc., Protein Engineering, 7:697-704 (1994)).Yet the antibody fragment in the context of the invention is not limited to the exemplary types of these antibody fragments.Can use any suitable can identify and in conjunction with the antibody fragment of expectation cell surface receptor or antigen.Antibody fragment further describes at for example Parham, J.Immunol, 131:2895-2902 (1983), the J.Immunol such as Spring, 113:470-478 (1974), and the Arch.Biochem.Biophys. such as Nisonoff, 89:230-244 (1960).Use any usability methods known in the art for example radioimmunoassay (RIA), ELISA, Western blotting, immuno-precipitation and competitive inhibition algoscopy, can measure antibody-antigen in conjunction with (referring to, for example, Janeway etc., above, and U.S. Patent Application Publication No. 2002/0197266A1).
In addition, described antibody can be chimeric antibody or its Fab." chimeric " refer to antibody comprise at least two available from or produce from least two kinds of different types of immunoglobulins or its segment (for example, two kinds of different immunoglobulins, as with human normal immunoglobulin's constant region of mouse immune globulin variable zone combination).Antibody also can be domain antibodies (dAb) or its Fab, for example, camelization (camelid) antibody (referring to, for example, Desmyter etc., Nature Struct.Biol, 3:752, (1996)), or shark antibody, for example, neoantigen receptor (IgNAR) (referring to, for example, Greenberg etc., Nature, 374:168 (1995) and Stanfield etc., Science, 305:1770-1773 (2004)).
Any applicable antibody can be used in the context of the present invention.For example monoclonal antibody J5 is Mus IgG2a antibody, it has specificity (Ritz etc. to CALLA (CALLA), Nature, 283:583-585 (1980)), the cell (for example, acute lymphoblastic leukemia cell) that can be used for targeted expression CALLA.Monoclonal antibody MY9 is Mus IgG1 antibody, its specific binding CD33 antigen (Griffin etc., Leukemia Res., 8:521 (1984)), but targeting is in the cell (for example, acute myeloid leukaemia (AML) cell) of expressing CD33.
Similarly, Monoclonal Antibody Against-B4 (also referring to do B4) is Mus IgG1 antibody, it is in conjunction with the CD19 antigen (Nadler etc. on the B cell, J.Immunol., 131:244-250 (1983)), the B cell or the diseased cells (for example, non Hodgkin lymphoma cell and chronic lymphatic leukemia cell) that can be used for targeted expression CD19.N901 is mouse monoclonal antibody, and its cell in conjunction with the neuroendocrine origin comprises CD56 (nerve cell adhesion molecule) antigen of finding on the small cell lung tumor, and it can be used for making in the conjugate drug targeting in the cell of neuroendocrine origin.J5, MY9 and B4 antibody are before they are used as the part of conjugate, preferably by resurfacing or humanization.The resurfacing of antibody or humanization for example are described in, Roguska etc., Proc.Natl.Acad.Sci.USA, 91:969-73 (1994).
In addition, monoclonal antibody C242 can be used for making the conjugate targeting in the tumor of expressing CanAg, for example colorectal cancer, cancer of pancreas, nonsmall-cell lung cancer and gastric cancer in conjunction with CanAg antigen (referring to for example, United States Patent (USP) 5,552,293).HuC242 is the humanization form (referring to for example, United States Patent (USP) 5,552,293) of monoclonal antibody C242.The hybridoma of producing HuC242 is preserved in ECACC, differentiates number to be 90012601.Application CDR engrafting method (referring to, for example, United States Patent (USP) 5,585,089,5,693,761 and 5,693,762) or the surface reconstruction technology (referring to, for example, United States Patent (USP) 5,639,641), can prepare HuC242.HuC242 can be used for making the conjugate targeting in the tumor cell of expressing CanAg antigen, for example, and colorectal cancer, cancer of pancreas, nonsmall-cell lung cancer and stomach cancer cell.
For targeting in ovarian cancer and prostate gland cancer cell, anti-MUC1 antibody can be used as the Cell binding agent in the conjugate.Anti-MUC1 antibody for example comprises, anti-HMFG-2 (referring to, for example, Taylor-Papadimitriou etc., Int.J.Cancer, 28:17-21 (1981)), hCTM01 (referring to, for example, van Hof etc., Cancer Res., 56:5179-5185 (1996)) and DS6.Anti-by using-prostate specific membrane antigen (PSMA) as Cell binding agent such as J591, prostate gland cancer cell also can by the conjugate targeting (referring to, for example, Liu etc., Cancer Res., 57:3629-3634 (1997)).In addition, use the antibody Herceptin, cancerous cell that can targeted expression Her2 antigen is breast carcinoma, carcinoma of prostate and ovarian cancer for example.The anti-IGF-IR antibodies of being combined with IGF-1 also can be used for conjugate.
Especially preferred antibody is Humanized monoclonal antibodies, and the example comprises huN901, huMy9-6, huB4, huC242, trastuzumab, bivatuzumab, sibrotuzumab and Rituximab (referring to, for example, United States Patent (USP) 5,639,641 and 5,665,357, U.S. Provisional Patent Application number 60/424,332 (they are relevant to United States Patent (USP) patent publication No. 2005/0118183A1), International Patent Application WO 02/16401, Pedersen etc., above, Roguska etc., above, Liu etc., above, Nadler etc., above, Colomer etc., Cancer Invest., 19:49-56 (2001), Heider etc., Eur.J.Cancer, 31A:2385-2391 (1995), Welt etc., J.Clin.Oncol, 12:1193-1203 (1994), and Maloney etc., Blood, 90:2188-2195 (1997)).Most preferably, antibody is huN901 Humanized monoclonal antibodies or huMy9-6 Humanized monoclonal antibodies.Other preferred antibody comprises CNTO95, huDS6, huB4 and huC242.Other Humanized monoclonal antibodies is known in the art, can use in conjunction with the present invention.
Although the Cell binding agent is preferably antibody, the Cell binding agent also can be the non-antibody molecule.Applicable non-antibody molecule for example comprises, interferon (for example, α, β or IFN-γ), lymphokine (for example, interleukin II (IL-2), IL-3, IL-4 or IL-6), hormone (for example insulin), somatomedin (for example, EGF, TGF-α, FGF and VEGF), colony stimulating factor (for example, G-CSF, M-CSF and GM-CSF (referring to, Burgess for example, Immunology Today, 5:155-158 (1984)), somatostatin and transferrins (referring to, for example, O'Keefe etc., JBiol.Chem., 260:932-937 (1985)).The GM-CSF of for example, being combined with myeloid cell can be used as Cell binding agent targeting in Acute Myeloid Leukemia Cells Contributing.In addition, the IL-2 of being combined with activating T cell can be used for prevention and transplants and to transfer plant and repel, and is used for the treatment of and prevents graft versus host disease, and be used for the treatment of acute T chronic myeloid leukemia.Epidermal growth factor (EGF) but targeting in scale cancer such as pulmonary carcinoma and head and neck cancer.Somatostatin can be used for targeting neuroblastoma cell and other tumor cell type.
Conjugate can comprise any suitable medicine, is generally cytotoxic agent." cytotoxic agent " used herein refers to cause cell death, lures any chemical compound of cell death or minimizing cell survival.Applicable cytotoxic agent for example comprises maytansinoid and maytansinoid analog, taxanes (taxoids), CC-1065 and CC-1065 analog and dolastatin and dolastatin analog.In the preferred embodiment of the invention, cytotoxic agent is maytansinoid, comprises maytansinol and maytansinol analog.The chemical compound that maytansinoid forms for suppressing microtubule has strong toxicity to mammalian cell.The example of the maytansinol analog that is fit to comprise aromatic ring with modification those and have those of modification in other position.Such maytansinoid for example is described in, United States Patent (USP) 4,256,746,4,294,757,4,307,016,4,313,946,4,315,929,4,322,348,4,331,598,4,361,650,4,362,663,4,364,866,4,424,219,4,371,533,4,450,254,5,475,092,5, in 585,499,5,846,545 and 6,333,410.
Example with maytansinol analog of modified aromatic ring comprises: (1) C-19-dechlorination (United States Patent (USP) 4; 256; 746) (P2 makes by LAH reduction ansamitocin (ansamytocin)); (2) C-20-hydroxyl (or C-20-demethyl)+/-C-19-dechlorination (United States Patent (USP) 4; 361; 650 and 4; 307; 016) (makes by using streptomycete (Streptomyces) or actinomycetes (Actinomyces) demethylation or using the LAH dechlorination) and (3) C-20-de-methoxy; the C-20-acyloxy (OCOR); +/-dechlorination (United States Patent (USP) 4; 294,757) (make by using acyl chlorine acidylate).
The example that has the maytansinol analog of modification in the position except aromatic ring comprises: (1) C-9-SH (United States Patent (USP) 4,424,219) is (by using H 2S or P 2S 5Make with the maytansinol reaction), (2) C-14-alkoxy methyl (de-methoxy/CH 2OR) (United States Patent (USP) 4,331,598), (3) C-14-methylol or acyloxy methyl (CH 2OH or CH 2OAc) (United States Patent (USP) 4,450,254) ((Nocardia) makes by Nocard's bacillus), (4) C-15-hydroxyl/acyloxy (United States Patent (USP) 4,364,866) (by making with streptomycete conversion maytansinol), (5) C-15-methoxyl group (United States Patent (USP) 4,313,946 and 4,315,929) (isolated from trewianudiflora (Trewia nudiflora)) (6) C-18-N-demethyl (United States Patent (USP) 4,362,663 and 4,322,348) (by with streptomycete the maytansinol demethylation being made) and (7) 4,5-deoxidation (United States Patent (USP) 4,371,533) (making by titanous chloride ./LAH reduction maytansinol).
In the preferred embodiment of the invention, conjugate is used the maytansinoid DM1 that contains mercaptan, is also referred to as N 2'-Tuo acetyl-N 2'-(3-sulfydryl-1-oxopropyl)-and maytansine, as cytotoxic agent.The structure of DM1 is represented by formula (I):
Figure BDA00002057608900131
In another preferred embodiment of the present invention, conjugate is used the maytansinoid DM4 that contains mercaptan, is also referred to as N 2'-Tuo acetyl-N 2'-(4-methyl-4-sulfydryl-1-oxo amyl group)-and maytansine, as cytotoxic agent.The structure of DM4 is represented by formula (II):
Figure BDA00002057608900141
Other maytansine can be used for for example comprising in the context of the present invention, carries the maytansinoid that contains mercaptan and disulphide that single or two alkyl replaces at the carbon atom that carries sulphur atom.Especially preferred is following maytansinoid: have (a) C-14 methylol, C-15 hydroxyl or C-20 demethyl degree of functionality in the C-3 position; (b) be carried the amino acid side chain of the acyl group acidylate that hinders sulfydryl; the acyl group carbon atom that wherein carries mercaptan functionality has one or two substituent groups, and described substituent group is CH 3, C 2H 5, have phenyl or heterocyclic aromatic base or a Heterocyclylalkyl of straight or branched alkyl or the alkenyl of 1 to 10 carbon atom, the cyclic alkyl with 3 to 10 carbon atoms or thiazolinyl, phenyl, replacement; wherein one of substituent group can be H further, and the acyl group straight chain that has at least three carbon atom length between carbonyl functionality and sulphur atom wherein.
Be used for the other maytansine of context of the present invention and comprise the chemical compound that is represented by formula (III):
Figure BDA00002057608900142
Wherein Y' representative
(CR 7R 8) l(CR 9=CR 10) pC ≡ C qA r(CR 5R 6) mD u(CR 11=CR 12) r(C ≡ C) sB t(CR 3R 4) n-CR 1R 2SZ, wherein R 1And R 2Independent separately is CH 3, C 2H 5, have phenyl or heterocyclic aromatic base or the Heterocyclylalkyl of straight chained alkyl or the alkenyl of 1 to 10 carbon atom, the side chain with 3 to 10 carbon atoms or cyclic alkyl or thiazolinyl, phenyl, replacement, and R wherein 2Also can be H,
Wherein A, B, D have the cycloalkyl of 3-10 carbon atom or cycloalkenyl group, simple or the aryl or heterocyclic aromatic base or the Heterocyclylalkyl that replace,
R wherein 3, R 4, R 5, R 6, R 7, R 8, R 9, R 11And R 12Independent separately is H, CH 3, C 2H 5, have phenyl or heterocyclic aromatic base or a Heterocyclylalkyl of straight chained alkyl or the alkenyl of 1 to 10 carbon atom, the side chain with 3 to 10 carbon atoms or cyclic alkyl or thiazolinyl, phenyl, replacement,
Wherein l, m, n, o, p, q, r, s and t independently are zero or 1 to 5 integer separately, and condition is that at least two among l, m, n, o, p, q, r, s and the t are non-vanishing in the time of any one time, and
Wherein Z is H, SR or COR, and wherein R is straight chained alkyl or the alkenyl with 1 to 10 carbon atom, the side chain with 3 to 10 carbon atoms or cyclic alkyl or thiazolinyl or aryl or heterocyclic aromatic base or Heterocyclylalkyl simple or that replace.
The preferred embodiment of formula (III) comprises wherein (a) R 1Be H, R 2For methyl and Z are H, (b) R 1And R 2For methyl and Z are H, (c) R 1Be H, R 2For methyl and Z are-SCH 3, and (d) R 1And R 2For methyl and Z are-SCH 3Formula (III) chemical compound.
The other maytansine of this class also comprises formula (IV-L), (IV-D) or (IV-D, L) represented chemical compound:
Figure BDA00002057608900151
Wherein Y represents (CR 7R 8) l(CR 5R 6) m(CR 3R 4) nCR 1R 2SZ,
R wherein 1And R 2Independent separately is CH 3, C 2H 5, have phenyl or heterocyclic aromatic base or the Heterocyclylalkyl of straight chained alkyl or the alkenyl of 1 to 10 carbon atom, the side chain with 3 to 10 carbon atoms or cyclic alkyl or thiazolinyl, phenyl, replacement, and R wherein 2Also can be H,
R wherein 3, R 4, R 5, R 6, R 7And R 8Independent separately is H, CH 3, C 2H 5, have phenyl or heterocyclic aromatic base or a Heterocyclylalkyl of straight chained alkyl or the alkenyl of 1 to 10 carbon atom, the side chain with 3 to 10 carbon atoms or cyclic alkyl or thiazolinyl, phenyl, replacement,
Wherein l, m and n independently are 1 to 5 integer separately, and n can be zero in addition,
Wherein Z is H, SR or COR, and wherein R is straight or branched alkyl or the alkenyl with 1 to 10 carbon atom, the cyclic alkyl with 3 to 10 carbon atoms or thiazolinyl or aryl or heterocyclic aromatic base or Heterocyclylalkyl simple or that replace, and
Wherein the maytansinoid of side chain is carried in the May representative at C-3, C-14 methylol, C-15 hydroxyl or C-20 demethyl.
Formula (IV-L), (IV-D) and (IV-D, L) preferred embodiment comprise wherein (a) R 1Be H, R 2Be methyl, R 5, R 6, R 7And R 8The H that respectively does for oneself, l and m 1, the n that respectively does for oneself is 0, and Z is H, (b) R 1And R 2Be methyl, R 5, R 6, R 7And R 8The H that respectively does for oneself, l and m 1, the n that respectively does for oneself is 0, and Z is H, (c) R 1Be H, R 2Be methyl, R 5, R 6, R 7And R 8The H that respectively does for oneself, l and m 1, the n that respectively does for oneself is 0, and Z is-SCH 3, or (d) R 1And R 2Be methyl, R 5, R 6, R 7And R 8The H that respectively does for oneself, l and m 1, the n that respectively does for oneself is 0, and Z is-SCH 3Formula (IV-L), the chemical compound of (IV-D) and (IV-D, L).
Preferably, cytotoxic agent is represented by formula (IV-L).
Preferred maytansine also comprises the chemical compound by formula (V) expression in addition:
Figure BDA00002057608900161
Wherein Y represents (CR 7R 8) l(CR 5R 6) m(CR 3R 4) nCR 1R 2SZ,
R wherein 1And R 2Independent separately is CH 3, C 2H 5, have phenyl or heterocyclic aromatic base or the Heterocyclylalkyl of straight chained alkyl or the alkenyl of 1 to 10 carbon atom, the side chain with 3 to 10 carbon atoms or cyclic alkyl or thiazolinyl, phenyl, replacement, and R wherein 2Also can be H,
R wherein 3, R 4, R 5, R 6, R 7And R 8Independent separately is H, CH 3, C 2H 5, have phenyl or heterocyclic aromatic base or a Heterocyclylalkyl of straight chained alkyl or the alkenyl of 1 to 10 carbon atom, the side chain with 3 to 10 carbon atoms or cyclic alkyl or thiazolinyl, phenyl, replacement,
Wherein l, m and n independently are 1 to 5 integer separately, and n can be zero in addition, and
Wherein Z is H, SR or COR, and wherein R has straight chained alkyl or the alkenyl of 1 to 10 carbon atom, the side chain with 3 to 10 carbon atoms or cyclic alkyl or thiazolinyl or aryl or heterocyclic aromatic base or Heterocyclylalkyl simple or that replace.
The preferred embodiment of formula (V) comprises wherein (a) R 1Be H, R 2Be methyl, R 5, R 6, R 7And R 8H respectively does for oneself; L and m respectively do for oneself 1; N is 0, and Z is H, (b) R 1And R 2Be methyl, R 5, R 6, R 7And R 8The H that respectively does for oneself, l and m respectively do for oneself 1; N is 0, and Z is H, (c) R 1Be H, R 2Be methyl, R 5, R 6, R 7And R 8The H that respectively does for oneself, l and the m l that respectively does for oneself, n is 0, and Z is-SCH 3, or (d) R 1And R 2Be methyl, R 5, R 6, R 7And R 8The H that respectively does for oneself, l and m are that 1, n is 0, and Z is-SCH 3Formula (V) chemical compound.
Further preferred maytansine comprises the chemical compound by formula (VI-L), (VI-D) or (VI-D, L) expression again:
Figure BDA00002057608900171
Y wherein 2Representative (CR 7R 8) l(CR 5R 6) m(CR 3R 4) nCR 1R 2SZ 2,
R wherein 1And R 2Independent separately is CH 3, C 2H 5, have phenyl or heterocyclic aromatic base or the Heterocyclylalkyl of straight chained alkyl or the alkenyl of 1 to 10 carbon atom, the side chain with 3 to 10 carbon atoms or cyclic alkyl or thiazolinyl, phenyl, replacement, and R wherein 2Also can be H,
R wherein 3, R 4, R 5, R 6, R 7And R 8Independent separately is H, CH 3, C 2H 5, have phenyl or heterocyclic aromatic base or a Heterocyclylalkyl of straight chain cyclic alkyl or the thiazolinyl of 1 to 10 carbon atom, the side chain with 3 to 10 carbon atoms or cyclic alkyl or thiazolinyl, phenyl, replacement,
Wherein l, m and n independently are 1 to 5 integer separately, and n can be zero in addition,
Z wherein 2Be SR or COR, wherein R is straight chained alkyl or the alkenyl with 1 to 10 carbon atom, the side chain with 3 to 10 carbon atoms or cyclic alkyl or thiazolinyl or aryl or heterocyclic aromatic base or Heterocyclylalkyl simple or that replace, and
Wherein May represents maytansinoid.
Preferred maytansine comprises the chemical compound by formula (VII) expression in addition:
Figure BDA00002057608900181
Y wherein 2'Representative
(CR 7R 8) l(CR 9=CR 10) p(C≡C) qA r(CR 5R 6) mDu(CR 11=CR 12) r(C≡C) sB t(CR 3R 4) n-CR 1R 2SZ 2，
R wherein 1And R 2Independent separately is CH 3, C 2H 5, have phenyl or heterocyclic aromatic base or the Heterocyclylalkyl of straight or branched alkyl or the alkenyl of 1 to 10 carbon atom, the cyclic alkyl with 3 to 10 carbon atoms or thiazolinyl, phenyl, replacement, and other R 2Also can be H,
Wherein A, B, D be independently of one another for having aryl or heterocyclic aromatic base or the Heterocyclylalkyl of the cycloalkyl of 3-10 carbon atom or cycloalkenyl group, simple or replacement,
R wherein 3, R 4, R 5, R 6, R 7, R 8, R 9, R 11And R 12Independent separately is H, CH 3, C 2H 5, have phenyl or heterocyclic aromatic base or a Heterocyclylalkyl of straight chained alkyl or the alkenyl of 1 to 10 carbon atom, the side chain with 3 to 10 carbon atoms or cyclic alkyl or thiazolinyl, phenyl, replacement,
Wherein l, m, n, o, p, q, r, s and t independently are zero or 1 to 5 integer separately, and condition is that at least two among l, m, n, o, p, q, r, s and the t are non-vanishing in the time of any one time, and
Z wherein 2For SR or-COR, wherein R is straight chained alkyl or the alkenyl with 1 to 10 carbon atom, the side chain with 3 to 10 carbon atoms or cyclic alkyl or thiazolinyl or aryl or heterocyclic aromatic base or Heterocyclylalkyl simple or that replace.
The preferred embodiment of formula (VII) comprises wherein R 1Be H and R 2Formula (VII) chemical compound for methyl.
Except maytansinoid, the cytotoxic agent that is used for conjugate can be the taxane or derivatives thereof.Taxane is a compounds of group, comprises cytotoxicity natural product paclitaxel
Figure BDA00002057608900191
With semisynthetic derivative docetaxel
Figure BDA00002057608900192
Both all are widely used in treatment of cancer.Taxane is the mitosis spindle toxic agent, suppresses the tubulin depolymerization, causes cell death.Although docetaxel and paclitaxel are medicines useful in the treatment of cancer, their anti-tumor activity is owing to they are restricted Normocellular non-specific toxicity.In addition, resemble the not enough conjugates that effectively is used for the Cell binding agent of paclitaxel and docetaxel chemical compound itself.
Preferred taxane for the preparation of peptide-cytotoxic conjugates is the taxane of formula (VIII):
Synthetic can be used for the method for the taxane in the context of the invention and make taxane be conjugated to for example method of antibody of Cell binding agent, be described in detail in United States Patent (USP) 5,416,064,5,475,092,6,340,701,6,372,738,6,436,931,6,596,757,6,706,708 and 6,716,821, and among the U.S. Patent Application Publication No. 2004/0024049A1.
Cytotoxic agent also can be the CC-1065 or derivatives thereof.The effective antitumor antibiotic of CC-1065 for from damp ear streptomycete (Streptomyces zelensis) culture broth, separating.CC-1065 external and normally used cancer therapy drug for example doxorubicin, methotrexate and vincristine compare, act on powerful about 1000 times (Bhuyan etc., Cancer Res., 42:3532-3537 (1982)).CC-1065 and analog thereof are described in United States Patent (USP) 5,585, in 499,5,846,545,6,340,701 and 6,372,738.The cytotoxicity potential of CC-1065 is relevant with its activity in conjunction with DNA or the intercalation of DNA with its Alkylating Activity.These two kinds of activity are attributed to the unitary part of this molecule.In this respect, Alkylating Activity is included in two pyrrolo-indole subunits that ring the third pyrrolo-indole (CPI) subunit and dna binding activity are attributed to CC-1065.
Several CC-1065 analog are known in the art, also can be used as cytotoxic agent in the conjugate (referring to, for example, Warpehoski etc., J Med.Chem., 31:590-603 (1988)).Developed a series of CC-1065 analog, wherein the CPI part is by ring propyl benzene diindyl (CBI) Partial Replacement (Boger etc., J.Org.Chem., 55:5823-5833 (1990), with Boger etc., Bioorg.Med.Chem.Lett, 1:115-120 (1991)).These CC-1065 analog keep the external high-effect of parent drug, and do not cause the mice delayed toxicity.As CC-1065, these chemical compounds are alkylating agent, and its covalent bond DNA ditch causes cell death.
Distribute by changing in the body to tumor locus through targeted delivery, can greatly improve the therapeutic efficiency of CC-1065 analog, cause the toxicity lower to non-target tissue, and therefore reduce general toxicity.For this purpose, produced the analog of CC-1065 and the conjugate of derivant and Cell binding agent, its special target in tumor cell (referring to, for example, United States Patent (USP) 5,475,092,5,585,499 and 5,846,545).These conjugates are at external general demonstration height target-specific cytotoxin, and in mice, show in people's tumor xenogeneic graft model anti-tumor activity (referring to, for example, Chari etc., Cancer Res., 55:4079-4084 (1995)).
The method of synthetic CC-1065 analog is described in detail in United States Patent (USP) 5,475,092,5,585,499,5,846,545,6,534,660,6,586,618 and 6,756,397 and U.S. Patent Application Publication No. 2003/0195365A1 in.
Medicine is the plain analog of methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine, melphalan, ametycin, chlorambucil, calicheamicin, tubule element (tubulysin) and tubule, times carcinomycin and doubly carcinomycin analog, dolastatin and dolastatin analog for example, also can be used in the context of the present invention.Doxorubicin and daunorubicin chemical compound (referring to, for example, United States Patent (USP) 6,630,579) also useful as drug.
Drug conjugate can make by in vitro method.In order to connect medicine or prodrug to antibody, use linking group.The linking group that is fit to is well-known in the art, comprises disulfide group, to the unsettled group of acid, to the group of photo-labile, to the unsettled group of peptidase with to the unsettled group of esterase.Preferred linking group is disulfide group.For example, use the disulfide exchange reaction between antibody and medicine or the prodrug can consist of conjugate.By intermediate carrier molecule serum albumin for example, drug molecule also can be connected with the Cell binding agent.
According to the present invention, by making difunctional cross-linking reagent and Cell binding agent reaction, thereby cause connector molecule and Cell binding agent covalently bound, the modified cells bonding agent." difunctional cross-linking reagent " used herein is for making the covalently bound any chemical part of Cell binding agent and medicine (medicine as described herein).In the preferred embodiment of the invention, the part of coupling part is by drug provision.In this respect, medicine comprises the coupling part, and it is the part of major connector molecule more, and the connector molecule is used for the Cell binding agent is connected with medicine.For example, in order to form maytansinoid DM1, the side chain of maytansine C-3 hydroxyl is modified, to have free sulfydryl (SH).This mercaptan form of maytansine can react with the Cell binding agent of modifying, and forms conjugate.Therefore, final connector is assembled by two kinds of components, and wherein a kind of component is provided by cross-linking reagent, and another kind of component is provided by the side chain of DM1.
Any suitable difunctional cross-linking reagent can use in conjunction with the present invention, as long as this connector reagent provides respectively the therapeutic (for example cytotoxicity) of medicine and Cell binding agent and the reservation of targeting characteristic.Preferably, the connector molecule connects medicine to the Cell binding agent, so that medicine and each other chemical coupling of Cell binding agent (for example, covalent bonding) by chemical bond (as mentioned above).Preferably, connecting reagent is the cleavable connector.More preferably, connector is cleavable under the temperate condition, is not namely affecting under the intracellular condition of pharmaceutically active.The example of applicable cleavable connector comprises the disulphide connector, to the unsettled connector of acid, to the connector of photo-labile, to the unsettled connector of peptidase with to the unsettled connector of esterase.The connector that comprises disulphide is the connector by generable disulfide exchange cleavable under the physiological condition.The connector of cleavable under acid pH to the unsettled connector of acid.For example, some intracellular cell such as endosome and lysosome have acid pH (pH 4-5), and the condition that provides is fit to cracking to the unsettled connector of acid.The connector of photo-labile is applicable to contact body surface and many body cavitys of light.In addition, infrared ray can infiltrate tissue.To the unsettled connector of peptidase can be used for inside and outside the cell lysis or extracellular some peptide (referring to, for example, Trouet etc., Proc.Natl.Acad.Sci.USA, 79:626-629 (1982), and Umemoto etc., Int.J.Cancer, 43:677-684 (1989)).
Preferably, medicine is connected with the Cell binding agent by disulfide bond.The connector molecule comprises the reactive chemical group that can react with the Cell binding agent.Reactive chemical group preferred and that the Cell binding agent reacts is N-succinimido ester and N-sulfosuccinic acylimino ester.In addition, the connector molecule comprises reactive chemical group, preferred dithio pyridine radicals, its can with the medicine formation disulfide bond that reacts.Especially preferred connector molecule for example comprises, 3-(2-pyridine disulfide group) propanoic acid N-succinimido ester (SPDP) (referring to, for example, Carlsson etc., Biochem.J, 173:723-737 (1978)), 4-(2-pyridine disulfide group) butanoic acid N-succinimido ester (SPDB) (referring to, for example, United States Patent (USP) 4,563,304), 4-(2-pyridine disulfide group) valeric acid N-succinimido ester (SPP) (referring to, for example, CAS registration number 341498-08-6) and United States Patent (USP) 6,913, reactive intersection-the connector of other that describe in 748 is introduced it in full at this, as a reference.
Although preferably the cleavable connector is used for the inventive method, non-cracking performance connector also can be used for producing above-mentioned conjugate.Non-cracking performance connector for can connect in the mode of stable covalency medicine for example maytansinoid, taxane or CC-1065 analog to any chemical part of Cell binding agent.Therefore, non-cracking performance connector can be resisted in fact the cracking that acid causes, the cracking that light causes, the cracking that peptidase causes, cracking and the disulfide bonds that esterase causes under medicine or Cell binding agent keep active condition.
[0062] the suitable cross-linking reagent of the non-cracking performance connector of formation is well-known in the art between medicine and Cell binding agent.The example of non-cracking performance connector comprises the dimaleoyl imino that has the N-succinimido ester that reacts with the Cell binding agent or N-sulfosuccinic acylimino ester moiety and react with medicine-or take the connector of halo acetyl group as the part on basis.Comprise the amino cross-linking reagent for basic part of maleimide and comprise 4-(maleimide amino methyl) cyclohexane-carboxylic acid N-succinimido ester (SMCC); " long-chain " analog N-succinimido-4-(N-maleimide amino methyl) of SMCC-cyclohexane extraction-1-carboxyl-(6-acylamino-alkyl caproate) (LC-SMCC); κ-maleimide aminoundecanoic acid N-succinimido ester (KMUA); γ-maleimide aminobutyric acid N-succinimido ester (GMBS); ε-maleimide aminocaproic acid N-hydroxy-succinamide ester (EMCS); meta-maleimide amino benzoyl-N-hydroxy-succinamide ester (MBS); N-(α-maleimide glycyl oxygen base)-succinimide ester (AMAS); succinimido-6-(β-maleimide aminopropan acylamino-) alkyl caproate (SMPH); 4-(p-maleimide aminophenyl)-butanoic acid N-succinimido ester (SMPB) and N-(p-maleimide aminophenyl)-isocyanates (PMPI).Comprise the halo acetyl group and comprise N-succinimido-4-(iodo acetyl group)-Aminobenzoate (SIAB), iodine acetic acid N-succinimido ester (SIA), bromoacetic acid N-succinimido ester (SBA) and 3-(acetyl bromide amino) propanoic acid N-succinimido ester (SBAP) for the cross-linking reagent of part on basis.
Other cross-linking reagent that lacks the non-cracking performance connector of forming of sulphur atom also can be used in the inventive method.Such connector can be produced by the part take dicarboxylic acids as the basis.The suitable alpha, omega-dicarboxylic acid that includes but not limited to general formula (IX) take dicarboxylic acids as the part on basis:
HOOC-X l-Y n-Z m-COOH
(IX)，
Wherein X is straight or branched alkyl, alkenyl or the alkynyl with 2 to 20 carbon atoms, Y is cycloalkyl or the cycloalkenyl group that carries 3 to 10 carbon atoms, Z is replacement or the unsubstituted aromatic group that carries 6 to 10 carbon atoms, or replacement or unsubstituted wherein hetero atom are selected from the heterocyclic radical of N, O or S, wherein l, m and n respectively do for oneself 0 or 1, and condition is that l, m and n all are not zero simultaneously.
[0064] many non-cracking performance connectors disclosed herein are described in detail in the Application No. 10/960,602, and it is corresponding to U.S. Patent Application Publication No. 2005/0169933A1.
Alternative ground, disclosed among the 163B1 as United States Patent (USP) 6,441, the reactive ester that medicine can at first be fit to by modifying to introduce and the Cell binding agent reacts.These contain the maytansinoid of activation connector part and the reaction of Cell binding agent, and the method for Cell binding agent maytansinoid conjugate another kind of generation cleavable or non-cleavable is provided.
Additional information about maytansinoid, the cytotoxic agent that comprises it, drug conjugate and related manufacturing processes is disclosed in Application No. 11/352,121 and Application No. 10/849, in 136, the latter is corresponding to U.S. Patent Application Publication No. 2004/0235840A1.
The following example further illustrates the present invention, certainly should by any way it be construed as limiting the scope of the invention.
The specific embodiment
Embodiment 1
Present embodiment is used the purification that the TFF proof is used the antibody of isodigeranyl functionalized modification reagent modification.
At 20 ℃, in the 50mM kaliumphosphate buffer that contains 50mM NaCl, 2mM EDTA and 5% ethanol (pH 7.5), huN901 monoclonal antibody (final concentration 8mg/ml) and 4-(2-pyridine radicals disulfide group) valeric acid N-succinimido ester (SPP, 5.6 times of molar excess) were hatched about 180 minutes together.In first group, use Sephadex TMThe G25F resin column makes column equilibration and eluting with the 50mM kaliumphosphate buffer (pH 6.5) that contains 50mM NaCl and 2mM EDTA, with the reactant mixture purification.In second group, use Pellicon XLTFF system (Millipore, Billerica, MA) purification reaction mixture, use 10,000 molecular weight and block film (Ultracel TMRCF regenerated cellulose film, Millipore, Billerica, MA) with antibody diafiltration (5 volumes) to 50mM potassium phosphate, 50mM NaCl (pH 6.5) and 2mMEDTA.Two kinds of samples were puted together 18 hours with DM1 (with respect to 1.7 times of molar excess of unconjugated connector) in containing the pH6.5 kaliumphosphate buffer that 50mM NaCl and final concentration are 3%DMA.
In two groups, measure the productive rate of combination modification and purification step with spectrophotometry de termination (wavelength 280nm).(it is 8,080M at 343nM place extinction coefficient by process to discharge pyridine-2-thioketone with dithiothreitol, DTT -1Cm -1), also measured the ratio of connector/antibody.Measure the ratio of medicine/antibody of puting together step with spectrophotometry de termination (wavelength 280nm and 252nm).The relevant micromolecule kind of SPP of removing by Hisep HPLC measurement in addition.
The data description that obtains is in table 1.
Table 1: the purification process of using the huN901 of G-25F and TFF modification relatively
Figure BDA00002057608900251
As shown in table 1, the application of TFF produces the drug conjugate product, and its quality is equivalent to non-adsorption charomatography (G25) method at least, but more convenient and be produced on a large scale.
Embodiment 2
Present embodiment is used the purification that the adsorption charomatography proof is used the antibody of isodigeranyl functionalized modification reagent modification.
In room temperature, in the 50mM kaliumphosphate buffer that contains 50mM NaCl, 2mM EDTA and 5% ethanol (pH 6.5), huB4 antibody was modified 120 minutes with 4-(2-pyridine radicals disulfide group) butanoic acid N-succinimido ester (SPDB, 5.4 times of molar excess).In first group, use as described in Example 1 Sephadex TMThe G25F resin is with the reactant mixture purification.In second group, reactant mixture is loaded into ceramic hydroxyapatite column (CHT, Bio-Rad Laboratories, Hercules, CA), in 12.5mM kaliumphosphate buffer (pH 6.5), make column equilibration also with 80mM kaliumphosphate buffer (pH 6.5) eluting.
In two groups, measure as described in Example 1 the ratio of productive rate and connector/antibody.First group of ratio with connector/antibody of 91% productive rate and 4.2.Second group of ratio with connector/antibody of 89% productive rate and 4.2.
At 20 ℃, in the 10mM sodium phosphate buffer that contains 2.7% sucrose and 5% ethanol (pH 7.5), CNTO95 antibody (final concentration 10mg/ml) was modified 120 minutes with 4-(2-pyridine radicals disulfide group) butanoic acid N-succinimido ester (SPDB, 4.5 times of molar excess).In first group, use Sephadex TMThe G25F resin is in containing the 12.5mM kaliumphosphate buffer of 12.5mM NaCl and 0.5mMEDTA (pH 6.6), with the reactant mixture purification.In second group, reactant mixture is loaded into SP Sepharose Fast Flow post (GEHealthcare, Piscataway, NJ), make column equilibration and use 50mM kaliumphosphate buffer (pH 7.5) eluting that contains 50mM NaCl with 10mM sodium phosphate buffer (pH 7.5).
In two groups, measure as described in Example 1 the ratio of productive rate and connector/antibody.First group of ratio with connector/antibody of 96% productive rate and 4.0.Second group of ratio with connector/antibody of 97% productive rate and 4.1.
The digital proof adsorption charomatography that obtains in the present embodiment can be used for the antibody that purification is modified by isodigeranyl functionalized modification reagent.
Embodiment 3
Present embodiment proof is at the pH beneficial effect that modified antibody and medicine are puted together more than 6.5.
In first experiment, CNTO95 antibody is modified and purification as described in Example 2.Then the antibody of modifying is divided into two groups.In first group, at 20 ℃, in the 12.5mM kaliumphosphate buffer of the pH 6.5 of the medicine that contains 12.5mMNaCl, 0.5mM EDTA, 3%DMA and 1.7 times of molar excess of every connector, put together.In second group, conjugation reaction is carried out for 7.5 times at pH.The antibody of puting together is through the NAP-10 column purification.
Measure the ratio of medicine/antibody of two groups.The data description that obtains is in table 2.
The ratio of the medicine/antibody of table 2:pH 6.5 and 7.5 conjugation reaction relatively
Figure BDA00002057608900261
Shown in the data of describing in the table 2, be conjugated in pH 7.5 than carrying out sooner at pH 6.5.
In second experiment, the huB4 Humanized monoclonal antibodies is by following modification: (a) with respect to the SPDB of 4.9 times of molar excess of antibody, or (b) with respect to the SPDB of 4.8 times of molar excess of antibody.Under two kinds of situations, room temperature was reacted total 120 minutes in 5% ethanol that contains 50mM potassium phosphate, 50mM potassium chloride and 2mM EDTA (pH 6.5).With sample (a) through Sephadex TMG25F resin column purification, described post is balance in the 50mM of pH 6.5 potassium phosphate, 50mM sodium chloride and 2mM EDTA.Sample (b) is equal to purification, except the chromatograph buffer is adjusted to pH 7.5.Room temperature is puted together two kinds of samples and DM4 (with respect in conjunction with 1.7 times of molar excess of connector) 18 hours in final concentration is 3% dimethyl acetylamide (DMA).
Therefore, sample (a) is puted together at pH 6.5, and sample (b) is puted together at pH 7.5.Then with these samples through Sephadex TMG25F resin column purification, described post is balance in the 9.6mM of pH 6.5 potassium phosphate and 4.2mM sodium chloride.Two kinds of samples are hatched 7 months at the most at 4 ℃, and the free drug that discharges is analyzed in the compartment of terrain.The data description that obtains is in table 3.
Table 3: free drug is the release from the sample that pH 6.5 and 7.5 puts together in time
Time (moon) PH 6.5 puts together PH 7.5 puts together
0 1.0 0.8
1.5 1.8 1.0
2.5 3.2 1.9
7 4.0 2.8
Shown in the data of describing in the table 3, with respect to the sample of puting together at pH 6.5 (a), the release of free drug from the sample (b) of puting together at pH 7.5 is slower in fact.Correspondingly, compare with the drug conjugate product for preparing at pH 6.5, the drug conjugate product for preparing at pH 7.5 demonstrates more stable with regard to free drug release in time.Demonstrate better medicine fusion in puting together also of pH 7.5 than puting together at pH 6.5, thereby the medicine that need to use still less.
Embodiment 4
The beneficial effect that the antibody that present embodiment proof is modified below 6.0 at pH and medicine are puted together.
At 20 ℃, in the 50mM kaliumphosphate buffer that contains 50mM NaCl, 2mM EDTA and 5% ethanol (pH 7.5), huN901 monoclonal antibody (final concentration 8mg/ml) and 4-(2-pyridine radicals disulfide group) valeric acid N-succinimido ester (SPP, 5.6 times of molar excess) were hatched about 180 minutes together.In first group, use Sephadex TMThe G25F resin column makes column equilibration and eluting with the 50mM sodium citrate buffer solution (pH 5.0) that contains 50mM NaCl and 2mM EDTA, with the reactant mixture purification.In second group, use Sephadex TMThe G25F resin column makes column equilibration and eluting with the 50mM kaliumphosphate buffer (pH 6.5) that contains 50mM NaCl and 2mM EDTA, with the reactant mixture purification.Room temperature is puted together two kinds of samples and DM4 (with respect to 1.7 times of molar excess of connector of combination) 3,19,25,48 and 120 hours in final concentration is 3% dimethyl acetylamide (DMA).
Therefore, first group of sample puted together in the 50mM sodium citrate buffer solution that contains 50mM NaCl and 2mM EDTA (pH 5.0), and second group of sample contains in the 50mM sodium phosphate buffer (pH 6.5) of 50mM NaCl and 2mM EDTA to be puted together.Then with these samples through Sephadex TMG25F resin column purification, described post 50mM kaliumphosphate buffer (pH 6.5) balance and the eluting that contains 50mM NaCl.
In two groups, (it is 8,080M at 343nM place extinction coefficient by process to discharge pyridine-2-thioketone with dithiothreitol, DTT -1Cm -1), measured the ratio of connector/antibody.Measure the ratio of medicine/antibody of puting together step with spectrophotometry de termination (wavelength 280nm and 252nm).
First group of ratio with connector/antibody of 4.3.Second group of ratio with 4.2 connectors/antibody.
The ratio of two groups of medicine/antibody in time is described in the table 4.
Table 4:DM1 mixes the speed among the huN901 that SPP modifies, for puting together the function of pH
Figure BDA00002057608900291
Be apparent that from the data described in the table 4, compare with the conjugate of when pH 6.5 puts together, making that the antibody by making modification and medicine are puted together the conjugate of making and reached higher and the bound drug of maintenance level more at pH 5.0 in the conjugation reaction process.Except increasing stability, the result shows to put together at pH 6.5 with the medicine of using same amount and compares, and has reached higher medicine/antibody horizontal when pH 5.0 puts together, thereby has shown more efficient medicinal application when pH 5.0.
In two groups, measured conjugate amount of monomer in time.The data description that obtains is in table 5.
Table 5: during huN901 that SPP modifies and DM1 put together, put together pH to the impact of conjugate single level
Figure BDA00002057608900292
Be apparent that from the data described in the table 5, compare with the conjugate of when pH 6.5 puts together, making that the antibody by making modification and medicine are puted together the conjugate of making at pH 5.0 and had higher levels of conjugate monomer.
Embodiment 5
Present embodiment is the proof benefit of puting together less than the antibody of 6 o'clock medicines and modification at pH further.
In room temperature, in 50mM kaliumphosphate buffer (pH 6.5), 50mM NaCl, 2mMEDTA and 5% ethanol, BIWA 4 antibody were modified 120-140 minute with SPP (molar excess of SPP is as shown in table 6).The modified antibodies warp of equal sample is NAP 25 column purifications separately, the described post buffer balance that multiple pH value (pH 4.6-6.5) arranged.PH 4.6-5.9 buffer is comprised of 35mM sodium citrate, 150mM sodium chloride and 2mM EDTA.PH 6.5 buffer are the PBS that contains 2mM EDTA.
In dimethyl acetylamide (DMA, final concentration 3%), the antibody of modifying is puted together with DM1 (with respect to 1.7 times of molar excess of connector) under various pH.After incubated at room 17-18 hour, the antibody sample of puting together carries out chromatography purification by NAP 25 posts with PBS (pH 6.5) balance.(it is 8,080M at 343nM place extinction coefficient by process to discharge pyridine-2-thioketone with dithiothreitol, DTT -1Cm -1), measured the ratio (L/A in the table 6) of connector/antibody.Measure the ratio of medicine/antibody of puting together step with spectrophotometry de termination (wavelength 280nm and 252nm).Measured conjugate monomer, high molecular weight species and low molecular weight species by SEC-HPLC, it is applied in the TSKG3000SWXL post of the middle balance of the 0.2M kaliumphosphate buffer (pH 7.0) that contains 0.2M potassium chloride and 20% isopropyl alcohol and development.
This analysis result data is described in the table 6.
Table 6: the drug conjugate product is with respect to the characteristic of pH
Figure BDA00002057608900301
The digital proof of describing in the table 6 with compare in puting together of pH 6.5, SPP modifies when pH 6.0 is following BIWA 4 and puting together of DM1 are high efficiency.When lower pH, reduced the specific final medicine/connector of antibody ratio requirement and the amount of medicine, the particularly amount of SPP connector and DM1 of reaching.In addition, when lower pH, the level of conjugate monomer, high molecular weight species and low molecular weight species is more optimized, and productive rate is improved.
Embodiment 6
The step of the antibody that present embodiment proof purification is modified optionally is removed.Medicine can add simultaneously with difunctional modification reagent or add in the time after a while.
After modifying reagent, add in the example of medicine, at 20 ℃, with the Humanized monoclonal antibodies CNTO95 of 20mg/mL concentration with difunctional modification reagent SPDB (SPDB is with respect to 4.6 times of molar excess of antibody) modification 120 minutes.Modifying buffer is the 44mM phosphate buffer (pH 7.5) that contains 5.3% sucrose and 5% ethanol.The modified antibodies of one equal sample is through Sephadex TMG25F resin purification (standard 4 step methods), containing the 12.5mM kaliumphosphate buffer of 12.5mM NaCl (pH 7.5) balance and eluting, making ultimate density in room temperature subsequently is that 10mg/mL modified antibodies and DM4 (medicine is with respect to 1.7 times of molar excess of connector of combination) are containing in the 12.5mM kaliumphosphate buffer (pH 7.5) of 12.5mM NaCl and 10%DMA and puted together 20 hours.The modified antibodies of the second equal sample does not carry out further purification and puts together immediately (3 step method) when modification reaction finished in 120 minutes.
Regulate protein and the buffer concentration of modification reaction mixture, with the modifying protein that produces 10mg/mL concentration and the 28mM potassium phosphate buffer compositions (pH 7.5) that contains 5.9mM NaCl and 2.7% sucrose.Then add DM4 (with respect to beginning SPDB 1.7 times of molar excess), regulate DMA to final concentration be 10%.In incubated at room after 20 hours, two kinds of equal samples are puted together antibody through Sephadex TMThe G25F resin purification, described resin is balance in the 10mM of pH 5.5 histidine and 10% sucrose.
(it is 8,080M at 343nM place extinction coefficient by process to discharge pyridine-2-thioketone with dithiothreitol, DTT -1Cm -1), measured the ratio of connector/antibody (L/A).Measure ratio and the productive rate of medicine/antibody (D/A) of puting together step with spectrophotometry de termination (wavelength 280nm and 252nm).Analyze the percent of having measured monomer by SEC-HPLC.Measured the percent of free drug the analysis of Hisep post by HPLC.The results are described in the table 7 of these analyses.
Table 7: the optional purification step of removing modified antibodies
Parameter 4 step methods 3 step methods
Beginning SPDB 4.6x 4.6x
L/A 4.1 Do not measure
D/A 3.9 4.0
Productive rate 79% 91％
The % monomer 95.8% 96.1％
The % free drug 2.4% 1.1％
Prove such as the result who describes in the table 7, in the context of the invention, can remove the step of the antibody that purification modifies.
Embodiment 7
The modification method of present embodiment proof antibody purification, described antibody are modified with isodigeranyl functionalized modification reagent and are then puted together with maytansinoid.
To use as described in Example 1 SPP (7 times of molar excess) to modify and at Sephadex TMThe huN901 antibody of purification and maytansinoid DM1 on the G25F resin (with respect to 1.7 times of molar excess of connector, being dissolved in dimethyl acetylamide (DMA), final concentration 3%) put together.
The first sample conjugate passes through the reference colour spectrometry at Sephadex TMOn the G25F resin in the phosphate buffered saline (PBS) (PBS, pH 6.5) purification.
The second conjugate sample is as described in Example 1 by Pellicon XL TFF system (Millipore, Billerica, MA) purification.
The 3rd conjugate sample application MEP Hypercell resin column purification, described post is balance in 50mM Tris (pH 8.0), and with 50mM sodium acetate (pH 4.0) eluting.
The 4th conjugate sample application UNOsphere S resin column purification, described post is balance in 50mM sodium phosphate (pH 6.5), and with 0.2M NaCl and 50mM sodium phosphate (pH 6.5) eluting.
The 5th conjugate sample application CHT resin column (Bio-Rad Laboratories, Hercules, CA) purification, described post is balance in 50mM sodium phosphate (pH 6.5), and with 0.3M NaCl and 50mM sodium phosphate (pH 6.5) eluting.
The 6th conjugate sample application SP Sepharose resin column purification, described post is balance in 35mM sodium citrate, 10mM sodium chloride (pH 5.0), and with 0.25M NaCl, 35mM sodium citrate (pH 5.0) eluting.
Be applied in the TSKG3000SW of balance and development in the 0.2M kaliumphosphate buffer that comprises 0.2M potassium chloride and 20% isopropyl alcohol of pH 7.0 XLResin column is measured the conjugate monomer by SEC-HPLC.Productive rate by will puting together antibody has been measured the productive rate of puting together step divided by the amount (at wavelength 280nm place spectrophotometry measurement) of the modified antibodies of puting together.
The results are described in the table 8 of these analyses.
Table 8: the comparison of puting together purification step
The conjugate sample Put together purification step Conjugate monomer % The productive rate % of step
The 1(contrast) The G25F resin 93.2 86
The 2(invention) TFF 92.8 85
The 3(invention) MEP Hypercell resin 94.5 74
The 4(invention) The UNOsphere resin 96.3 81
The 5(invention) The CHT resin 97.9 72
The 6(invention) SP Sepharose resin 95.1 81
Result in the table 8 shows, all purification process of the present invention (2-6 group) all obtain the productive rate that obtains similar in appearance to contrast method (the 1st group).Every kind of chromatographic process of invention has produced the raising of conjugate single level and has expanded on a large scale easily and produce.
Except CHT (ceramic hydroxyapatite), under similar chromatography condition, also can use CFT (ceramic fluor-apatite).Alternative ground, the two all can be used for the mode of non-absorption CHT and CFT resin, so that the product (in fact monomer conjugate) of expectation do not keep by this resin, and keeps high molecular weight species, thereby with the product separation of expectation.
Although the standard buffer/solvent compositions that is used for puting together comprises 3%DMA, 50mM potassium phosphate, 50mM NaCl and 2mM EDTA, pH 6.5 (used such as embodiment 1), but other component and chromatography steps more as herein described are more compatible and other benefit is provided with respect to standard method.For example, puting together can be at 3%DMA, 12.5mM potassium phosphate, 12.5mMNaCl and 0.5mM EDTA(pH 6.5) in carry out.Under these conditions, the amount that the amount that DM4 mixes in huB4 antibody is mixed with respect to connector is than under the standard conditions high about 10%.In addition, these conditions are more suitable for being loaded into resin for example on cation exchange and the CHT resin.
All lists of references that this paper quotes comprise publication, patent application and patent, all are hereby incorporated by, and are indicated separately and specifically as each list of references to be hereby incorporated by, and are incorporated herein the full text of each list of references.
(especially in the context in following claim) application term " a " refers to similar with " the " with " an " in describing context of the present invention, all should be interpreted as encompasses singular and plural number, unless otherwise indicated herein or obviously contradict with context.Term " comprises ", " having ", " comprising " and " containing ", all should be interpreted as unless otherwise noted open-ended term (namely meaning " including but not limited to ").Unless the value scope that this paper enumerates is otherwise indicated herein, only be intended to drop on as indivedual expressions the stenography method of each independent value of this scope, each independent value is merged in the description, as this paper individually enumerates.All methods as herein described can be carried out with any suitable order, unless otherwise indicated herein or obviously contradict with context in addition.Application any and all examples or exemplary language (for example, " for example ") provided herein only is intended to explain the present invention better and does not form limitation of the scope of the invention, unless stated otherwise.The key element that any linguistic interpretation in the description should not protected for any failed call of expression is that enforcement is essential to the invention.
This paper describes the preferred embodiments of the invention, comprise enforcement known for inventor best way of the present invention.Those of ordinary skills are after reading above-mentioned explanation, and it is obvious that the variation of those preferred embodiments can become.The inventor expects the such variation of application when the technical staff is suitable, and the mode beyond the inventor plans clearly to describe with this paper is implemented the present invention.Therefore, the present invention includes applicable allowed by law all modifications and equivalent at the theme described in these claims.And above-mentioned key element all comprises in the present invention with any combination that it might change, unless otherwise indicated herein or obviously contradict with context in addition.

Claims (30)

1. the method for Dispersal risk-cytotoxic agent conjugate, the method comprises the following steps:
(a) with difunctional cross-linking reagent contact antibody, so that connector and antibody are covalently bound, thereby prepare the first mixture, this mixture comprises the antibody that combines connector on it,
(b) by being about 4 to about 9 solution, its antibody that combines connector and cytotoxic agent to be reacted at pH, the antibody that combines connector on it is puted together, to prepare the second mixture, this mixture comprises (i) by the antibody of connector and cytotoxic agent chemical coupling, (ii) free cell toxic agents and the byproduct of reaction that (iii) produces during the step (b), and
(c) make the second mixture stand tangential flow filtration, selective precipitation, adsorption filtration, adsorption charomatography or its combination, with purification from other composition of the second mixture by the antibody of connector and cytotoxic agent chemical coupling, thereby second mixture of the antibody that passes through connector and cytotoxic agent chemical coupling of preparation purification;
Wherein said the first mixture is without undergoing purification.
2. the process of claim 1 wherein that described adsorption charomatography is selected from hydroxyapatite chromatography method, dewatering electric charge inducing color chromatogram method, hydrophobic interaction chromatography, ion exchange chromatography, hybrid ion exchange chromatography, immobilized metal affinity chromatography, dye ligand chromatography, affinity chromatography, reverse-phase chromatography and combination thereof.
3. the process of claim 1 wherein that the pH of the solution in the step (b) is about 4 to about 6.0.
4. the process of claim 1 wherein that the pH of the solution in the step (b) is about 6.5 to about 9.
5. the process of claim 1 wherein that the solution in the step (b) comprises sucrose.
6. the process of claim 1 wherein that the solution in the step (b) comprises buffer agent, it is selected from citrate buffer agent, acetate buffer, succinate buffer agent and phosphate buffer.
7. the method for Dispersal risk-cytotoxic agent conjugate, the method comprises the following steps:
(a) with difunctional cross-linking reagent contact antibody, so that connector and antibody are covalently bound, thereby prepare the first mixture, this mixture comprises the antibody that combines connector on it,
(b) make the first mixture stand selective precipitation, adsorption filtration or adsorption charomatography, thereby combine the first mixture of the antibody of connector on its of preparation purification,
(c) by being about 4 to about 9 solution, its antibody that combines connector and cytotoxic agent to be reacted at pH, the antibody that combines connector on it is puted together, to prepare the second mixture, this mixture comprises (i) by the antibody of connector and cytotoxic agent chemical coupling, (ii) free cell toxic agents and (iii) byproduct of reaction, and
(d) make the second mixture stand tangential flow filtration, selective precipitation, adsorption filtration or adsorption charomatography, with purification from other composition of the second mixture by the antibody of connector and cytotoxic agent chemical coupling, thereby second mixture of the antibody that passes through connector and cytotoxic agent chemical coupling of preparation purification.
8. the method for claim 7 is wherein used tangential flow filtration in step (d).
9. the method for claim 7 is wherein used adsorption charomatography in step (b), and uses tangential flow filtration in step (d).
10. the method for Dispersal risk-cytotoxic agent conjugate, the method comprises the following steps:
(a) with difunctional cross-linking reagent contact antibody, so that connector and antibody are covalently bound, thereby prepare the first mixture, this mixture comprises the antibody that combines connector on it,
(b) make the first mixture stand tangential flow filtration, selective precipitation, adsorption filtration or adsorption charomatography, thereby combine the first mixture of the antibody of connector on its of preparation purification,
(c) by being about 4 to about 9 solution, its antibody that combines connector and cytotoxic agent to be reacted at pH, the antibody that combines connector on it is puted together, to prepare the second mixture, this mixture comprises (i) antibody by connector and cytotoxic agent chemical coupling, (ii) free cell toxic agents and (iii) byproduct of reaction, and
(d) make the second mixture stand selective precipitation, adsorption filtration or adsorption charomatography, with purification from other composition of the second mixture by the antibody of connector and cytotoxic agent chemical coupling, thereby the second mixture of the antibody that passes through connector and cytotoxic agent chemical coupling of preparation purification
Condition is, if stand tangential flow filtration at the first mixture described in the step (b), so at the second mixture described in the step (d) without undergoing adsorption charomatography.
11. the method for claim 10 is wherein used adsorption charomatography in step (b) with (d).
12. any one method of claim 7～11, wherein said adsorption charomatography is selected from hydroxyapatite chromatography method, dewatering electric charge inducing color chromatogram method, hydrophobic interaction chromatography, ion exchange chromatography, hybrid ion exchange chromatography, immobilized metal affinity chromatography, dye ligand chromatography, affinity chromatography, reverse-phase chromatography and combination thereof.
13. any one method of claim 7～11, wherein the pH of the solution in the step (c) is about 4 to 6.0.
14. any one method of claim 7～11, wherein the pH of the solution in the step (c) is 6.5 to about 9.
15. any one method of claim 7～11, wherein the solution in the step (c) comprises sucrose.
16. any one method of claim 7～11, wherein the solution in the step (c) comprises buffer agent, and it is selected from citrate buffer agent, acetate buffer, succinate buffer agent and phosphate buffer.
17. any one method of claim 1～11, wherein said antibody is monoclonal antibody.
18. any one method of claim 1～11, wherein said antibody is Humanized monoclonal antibodies.
19. any one method of claim 1～11, wherein said antibody is chimeric antibody.
20. any one method of claim 1～11, wherein said antibody is selected from huN901, huMy9-6, huB4, huC242, trastuzumab, bivatuzumab, sibrotuzumab, CNTO95, huDS6 and Rituximab.
21. any one method of claim 1～11, wherein said cytotoxic agent is selected from maytansinoid, taxanes and CC1065.
22. the method for claim 21, wherein said cytotoxic agent are maytansinoid.
23. the method for claim 22, wherein said maytansinoid comprises mercapto.
24. the method for claim 23, wherein said maytansinoid are N 2'-Tuo acetyl-N 2'-(3-sulfydryl-1-oxopropyl)-maytansine.
25. the method for claim 23, wherein said maytansinoid are N 2'-Tuo acetyl-N 2'-(4-methyl-4-sulfydryl-1-oxo amyl group)-maytansine.
26. any one method of claim 1～11, wherein said antibody is through chemical bond and described cytotoxic agent chemical coupling, and described chemical bond is selected from disulfide bond, to the unsettled key of acid, to the key of photo-labile, to the unsettled key of peptidase, thioether bond with to the unsettled key of esterase.
27. any one method of claim 1～11, wherein said difunctional cross-linking reagent is selected from 3-(2-pyridine disulfide group) propanoic acid N-succinimido ester; 4-(2-pyridine disulfide group) butanoic acid N-succinimido ester; 4-(2-pyridine disulfide group) valeric acid N-succinimido ester; 4-(maleimide amino methyl) cyclohexane-carboxylic acid N-succinimido ester; N-succinimido-4-(N-maleimide amino methyl)-cyclohexane extraction-1-carboxyl-(6-acylamino-alkyl caproate); κ-maleimide aminoundecanoic acid N-succinimido ester; γ-maleimide aminobutyric acid N-succinimido ester; ε-maleimide aminocaproic acid N-hydroxy-succinamide ester; meta-maleimide amino benzoyl-N-hydroxy-succinamide ester; N-(α-maleimide glycyl oxygen base)-succinimide ester; succinimido-6-(β-maleimide aminopropan acylamino-) alkyl caproate; 4-(p-maleimide aminophenyl)-butanoic acid N-succinimido ester; N-(p-maleimide aminophenyl)-isocyanates; N-succinimido-4-(iodo acetyl group)-Aminobenzoate; iodine acetic acid N-succinimido ester; bromoacetic acid N-succinimido ester and 3-(acetyl bromide is amino) propanoic acid N-succinimido ester.
28. any one method of claim 1～11, wherein said difunctional cross-linking reagent is 4-(2-pyridine disulfide group) butanoic acid N-succinimido ester.
29. any one method of claim 1～11, wherein said difunctional cross-linking reagent is 4-(2-pyridine disulfide group) valeric acid N-succinimido ester.
30. any one method of claim 1～11, wherein said difunctional cross-linking reagent is 4-(maleimide amino methyl) cyclohexane-carboxylic acid N-succinimido ester.
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